The present invention relates to novel screening methods which enable the selection of neurokinin-1 (NK-1) receptor antagonist compounds (e.g., a substance P receptor antagonist) which do not possess significant inhibitory potency towards cytochrome P450 enzymes, in particular, CYP2D6. The present invention also relates to a method of generating a pharmacophore model of NK-1 receptor antagonist compounds which do not possess significant inhibitory potency towards CYP2D6. The invention also relates to methods for the discovery of molecules that are NK-1 receptor antagonist compounds which do not possess significant inhibitory potency towards the CYP2D6 enzyme. The invention also relates to pharmaceutical compositions comprising a NK-1 receptor antagonist compound that does not possess significant inhibitory potency towards CYP2D6 as identified by methods of the invention. The invention further relates to the uses of a NK-1 receptor antagonist compound identified by the methods of the invention for the manufacture of medicaments and for the treatment of a condition, a disorder or a disease in a mammal for which such an NK-1 antagonist receptor compound is therapeutically useful.
In the field of drug development, the developer of pharmaceutical substances must investigate the potential for clinically significant negative drugxe2x80x94drug interactions in the situations where more than one drug may be co-administered to a patient. There is a also significant need in the field of drug development to ascertain the potential existence of these drugxe2x80x94drug interactions prior to commencing any investigation into drug development. The identification of the strong potential usefulness of a chemical compound as early as possible in vitro saves considerable investment of time and resources.
Antagonists for the human NK-1 receptor are likely to be therapeutically useful in treating nausea, asthma, migraine, arthritis, post-operative pain (Takeuchi et al., J. Med. Chem., 41: 3609-3623, 1998) and depression (Kramer et al., Science, 281: 1640-1645, 1998). It would therefore be advantageous to be able to ascertain whether or not a useful therapeutic compound will inhibit any drug metabolizing enzymes involved in the clearance of other co-administered pharmaceuticals. There is a critical need for efficient, rapid and reliable methods to select promising candidates in drug discovery that demonstrate potency towards the NK-1 receptor, but not towards drug metabolizing enzymes, such as the cytochrome P450 series.
Drugxe2x80x94drug interactions involving cytochrome P450 enzymes (CYPs) are an important factor in the question of whether a new chemical entity will survive through to the development stage. CYPs are members of a large superfamily of heme-thiolate proteins involved in the metabolism of endobiotics and xenobiotics across eukaryotes and procaryotes. (Nelson et al., Pharmacogenetics, 6: 1-42, 1996). The clinical relevance of CYPs are their central role in drug metabolism. They are present in all human tissues and may be inhibited by the co-administration of competing xenobiotics of the same enzyme. (Wrighton et al., Toxicologic Pathology, 23: 199-208, 1995). Much less is known about the endogenous functions of CYPs, except for their role in steroid metabolism and recent postulations about their roles in neurotransmitter metabolism (Hiroi et al., Biochem. Biophys. Res. Commun., 249: 838-43, 1998) and signaling pathways (Chan et al., Proc. Natl. Acad. Sci., 95: 10459-10464, 1998). The current understanding of the structural requirements of the CYP active site are presently limited to homology models using P450CAM, P450TERP and P450eryF. (Lewis et al., Xenobiotica, 27: 319-340, 1997).
One of the human CYPs, CYP2D6, has been well studied. The polymorphic enzyme, CYP2D6, represents only approximately 1.5% of the total human hepatic P450 (Shimada et al., J. Pharmacol. and Exp. Pharmacol., 270: 414-423, 1994), yet participates in the metabolism of over 30% of clinically prescribed drugs (Lewis et al., supra.) including many which have a narrow therapeutic index. (Spatzenegger and Jaeger, Drug Metabolism Reviews, 27: 397-417, 1995; Wu et al., Biochem. Pharmacol., 53: 1605-1612, 1997; Lewis et al., supra.). The clinical relevance of CYP2D6 is notable as approximately 7% of Caucasians are poor metabolizers of CYP2D6 substrates while 1% are ultrarapid metabolizers of CYP2D6 substrates. (Brosen, Ther. Drug Monitoring, 18: 393-396, 1996). If a drug, which is an inhibitor of CYP2D6, is co-administered with a drug which has CYP2D6-mediated biotransformation as its major clearance mechanism, there is the potential danger of the occurrence of a clinically hazardous event depending on the concentration of the drugs at the enzymes in question.
Therapeutic drug monitoring of CYP2D6 modulators is costly and impacts on health care costs; thus minimizing interactions with CYP2D6 is advantageous to the patient and the entire health care system. Ultimately, knowledge of CYP2D6 inhibitory potential may impact on whether a drug will be co-administered with drugs which are known substrates of CYP2D6. Accordingly, the ability to predict the likelihood of a molecule being a CYP2D6 inhibitor early in the discovery process allows for the more efficient synthesis of suitable candidate molecules without the structural features which cause undesirable inhibition. A means of screening for drugs which do not have significant interaction with the CYP2D6 enzyme is therefore desirable.
Present technologies have centered around the use of in vitro testing using human liver microsomes or recombinant CYPs and a known catalytic probe for CYP2D6 such as bufuralol. However, the possible volume of in vitro studies is often limited by equipment and materials cost, incubation volume and the rate of analytical determination. Alternatives to in vitro techniques as a preliminary screen, enabling the selection of compounds for later study in vitro, would need to be fast, cost effective and reliable.
One such alternative, embraced by the present invention, is the use of computational quantitative structure activity relationship (QSAR) modeling techniques as a screening device for inhibitory potency towards CYP2D6. In the past, computational techniques have led to the production of some computer generated substrate templates, pharmacophores as well as homology models of the active site of CYPs. A pharmacophore model generated by SYBYL has been derived from CYP2D6 inhibitors of bufuralol 1xe2x80x2-hydroxylation as a selective probe for generating Ki values. (Strobl et al., J. Med. Chem., 36:1136-1145, 1993). This inhibitor pharmacophore model suggested that a positive charge on a nitrogen atom and a flat hydrophobic region extending to 7.5 xc3x85 virtually perpendicular along the N-H axis are requirements for inhibitory activity. (Strobl et al., Id.). Very recently, attempts at pharmacophore modeling of diverse inhibitors using CATALYST(trademark) have been described which were used prospectively or retrospectively to predict inhibitor binding affinity for CYP2D6 (Ekins et al., Pharmacogenetics, 9: 477-489, 1999).
To date, however, closely related molecules from a single therapeutic class have not been used to model the CYP2D6 active site from the point of view of inhibitory activity. There have only been QSAR models from other CYPs generated using a single therapeutic class of compounds, such as quinolones for CYP1A2 (Fuhr et al., Mol. Pharmacol., 43: 191-199, 1993) or warfarin analogs for CYP2C9 (Jones et al., Drug Metabolism and Disposition, 24: 1-6, 1996).
Approaches towards modeling the common features of substrates and inhibitors of human CYPs in general from data generated in vitro have been recently shown using a CATALYST(trademark) pharmacophore approach (Ekins et al., supra; Ekins et al., J. Pharmacol. and Exp. Ther., 288: 21-29, 1999; Ekins et al., J. Pharmacol. and Exp. Ther., 290: 429-438, 1999; Ekins et al., J. Pharmacol. and Exp. Ther., 291: 424-433, 1999), comparative molecular field analysis (COMFA) (Jones et al., supra), and a molecular descriptor method (Bravi and Wikel, in press, Quant. Struct. Act. Rel., 2000). Two of these 3D-quantitative structure activity relationship (3D-QSAR) techniques have also been used to predict a test set of molecules absent from the training set (Ekins et al., J. Pharmacol. and Exp. Ther., 288: 21-29 (1999)). The utility of predictive CYP2D6 inhibitor pharmacophores had been recently shown in comparison to Ki (apparent) data (Ekins et al., Pharmacogenetics, 9: 477-489, 1999) where models generated with the CATALYS(trademark) program exhibited reasonable success in the prediction of Ki (apparent) values for molecules in a test set, outside of the training set.
With respect to NK-1 receptor antagonists, the only pharmacophores for such antagonists had been generated with those same antagonist compounds (Cascieri et al., Mol. Pharmacol., 47: 660-665 (1995); Jacoby et al., J. Receptor and Signal Transfer, 17: 855-873 (1997); Takeuchi et al., J. Med. Chem., 41: 3609-3623 (1998)). Site-directed mutagenesis studies have suggested a number of amino acids important for binding NK-1 antagonists (see, Underwood et al., Chem. Biol., 1: 211-221 (1994)). Similarly, structure alignments have suggested likely amino acids important within the receptor for binding molecules with either nitrogens in a quinuclidine ring or piperazine ring, acyclic amino ether, and N-acyl-tryptophan moieties (Cascieri et al., supra). This suggested accommodation of structural diversity may allow the synthesis of NK-1 antagonists without potent CYP2D6 interaction. Most recently a pharmacophore model for 30 non-peptidic and peptidomimetic NK-1 antagonists suggested two main structural fragments, one with at least two off-set stacked aromatic rings and the other with features necessary for hydrophobic, H-bond and salt bridge interactions (Jacoby et al., supra). Molecular structures were also docked in the receptor models to show how water soluble peptidomimetics protrude outside of the binding site. In contrast, a CoMFA model of 72 NK-1 antagonists refuted the stacked conformation of the two aromatic groups as this produced a less predictive model when assessed using a test set of 18 molecules (Takeuchi et al., supra).
The pharmacophore model of the present invention for the CYP2D6 inhibitory activity of NK-1 receptor antagonist compounds makes possible the screening, discovery and selection of molecules that possess the desired activity with respect to the NK-1 receptor without having undesired interaction with the CYP2D6 enzyme.
The present invention is directed to a method of generating a pharmacophore model for the CYP2D6 inhibitory potency of NK-1 receptor antagonist compounds comprising the steps of
(i) generating a set of three-dimensional conformers for each of the compounds in a training set comprising five or more NK-1 receptor antagonist compounds;
(ii) correlating each of the compounds of said training set with an observed value for CYP2D6 inhibitory potency;
(iii) generating from the conformers generated in step (i) a set of one or more pharmacophore test models, each said pharmacophore test model comprising three or more CYP2D6 enzyme active site features selected from the group consisting of the hydrogen bond donor feature, the hydrogen bond acceptor feature, the hydrophobic region feature, the ionizable region feature and the ring aromatic feature, arranged in three-dimensional space;
(iv) calculating the CYP2D6 inhibitory potency for each conformer generated in step (i) towards each of the pharmacophore test models generated in step (iii);
(v) calculating the total cost (or goodness of fit) for each pharmacophore test model; and
(vi) choosing the lowest cost (or best fit) pharmacophore test model as the pharmacophore model.
Preferably, each of the steps of the methods of the invention are carried out using molecular modeling software, more preferably one such as CATALYST(trademark) version 4 (Molecular Simulations, Inc., San Diego, Calif.), or other modeling programs known to those of skill in the art.
The term xe2x80x9ctraining set,xe2x80x9d as used herein, refers to the set of compounds used to build the pharmacophore model that possess known CYP2D6 inhibitory activity. The training set of NK-1 receptor antagonist compounds in step (i) are preferably chosen from known NK-1 antagonist compounds with CYP2D6 inhibitory activity IC50 values which span at least three orders of magnitude, more preferably from approximately 0.01 xcexcM to 250xcexcM. One preferred method uses a training set of at least 18 compounds, and most preferably, at least 26 compounds. Another preferred method uses training sets of compounds selected from those listed in Tables I and II. Further, the number of conformers in step (i) is preferably limited to 175 conformers, more preferably 255 conformers, with a potential energy range of 50 Kcal/mole, preferably 35 Kcal/mole, most preferably 10 Kcal/mole.
The term xe2x80x9cpharmacophore test modelxe2x80x9d as used herein, refers to a best guess (whether random or based upon a composite skewed in favor of the compounds in the training set exhibiting a high degree of CYP2D6 inhibitory potency) for the three-dimensional orientation of a set of features which describe the physical, chemical and/or electronic environment of the active site of the CYP2D6 enzyme, said features comprising, e.g., the hydrogen bond donor feature, the hydrogen bond acceptor feature, the hydrophobic region feature, the ionizable region feature and the ring aromatic feature. Preferably, in step (iii), at least ten pharmacophore test models are generated.
In step (iv), the calculation of the CYP2D6 inhibitory potency for each of the conformers of the compounds towards a particular pharmacophore test model is preferably performed via a xe2x80x9cfast-fitxe2x80x9d algorithm which finds the optimum fit of the particular conformer of a compound to a particular pharmacophore test model without performing an energy minimization on the conformers of the compound. The calculated CYP2D6 inhibitory potency may be calculated by means of a linear regression equation, among other techniques, which correlates the input parameters, i.e., the observed CYP2D6 inhibitory potency of a particular compound, with the features present in that compound. Solving the equation for a particular conformer in a particular pharmacophore test model yields a calculated inhibitory potency value. Preferably the CYP2D6 inhibitory potency is based upon observed IC50 values.
The total xe2x80x9ccostxe2x80x9d (or goodness of fit) for each pharmacophore test model in step (v) is calculated from the deviation between the estimated CYP2D6 inhibitory activity and the observed activity for each compound combined with the number of pharmacophore features in the pharmacophore test model. The pharmacophore test model with the lowest xe2x80x9ccostxe2x80x9d (or deviation between estimated and observed activity) is the model that is selected as it generally possesses features representative of all of the generated pharmacophore test models. Preferably, the observed CYP2D6 activity is given by the IC50 values measured in vitro of the inhibition of recombinant CYP2D6 enzyme activity in the presence of a known substrate, such as in the bufuralol l""hydroxylase assay. However, measured Ki or percent inhibition values may also be used as indicators of observed activity.
The present invention also relates to a method for screening an NK-1 receptor antagonist compound for significant inhibitory potency towards CYP2D6 comprising the steps of
(i) finding the optimum fit of the NK-1 antagonist compound to the pharmacophore model of the present invention;
(ii) calculating the CYP2D6 inhibitory potency for the NK-1 antagonist compound.
Another preferred method of the present invention for generating a pharmacophore model for the CYP2D6 inhibitory potency of NK-1 antagonist compounds comprises the step of
(i) correlating the chemical features of a training set of NK-1 receptor antagonist compound conformers with a set of two- and/or three-dimensional descriptors for the active site of the CYP2D6 enzyme;
(ii) generating an equation relating the observed CYP2D6 inhibitory potency of the training set of NK-1 antagonist compounds to a set of generated two- and/or three-dimensional descriptors for the NK-1 antagonist compound.
The pharmacophore model in this instance is in the form of an equation. Preferably, each of the steps of the methods of the invention are carried out using molecular modeling software, more preferably one such as CERIUS(trademark) (Molecular Simulations, Inc., San Diego, Calif.), or other modeling programs known to those of skill in the art.
Preferably, the steps of this method may be carried out using the set of two- and/or three-dimensional descriptors for a compound molecule found in the 3D-QSAR functionality of CERIUS2(trademark). Step (ii) of the method is preferably carried out using a genetic function approximation (GFA) equation. However, one may also use principle component analysis or partial least squares to correlate descriptors and activity. In addition, there are other regression techniques (linear or non-linear) available and known to those of skill in the art to perform this correlation.
The present invention is also directed to a method for screening NK-1 receptor antagonist compounds for CYP2D6 inhibitory potency comprising the steps of
(i) generating the two- and/or three-dimensional descriptors for an NK-1 antagonist compound;
(ii) inputting said two- and/or three-dimensional descriptors into a pharmacophore model equation relating the measured CYP2D6 inhibitory potency of a training set of NK-1 antagonist compounds to a set of two- and/or three-dimensional descriptors generated for those NK-1 antagonist compounds; and
(iii) solving said equation for the inhibitory activity of the NK-1 antagonist compound corresponding to the generated three-dimensional descriptors of step (i).
Steps (i) through (iii) are preferably carried out using a software program, e.g., CERIUS2(trademark), among others, known to those of skill in the art.
The present invention is also directed to a pharmacophore model for the CYP2D6 inhibitory potency of NK-1 antagonist compounds generated in accordance with the methods of the present invention. The pharmacophore model of the invention is preferably three-dimensional and comprises a set of features, each of which is defined by Cartesian coordinates, x, y and z, which represent the centroid of the feature, and a vector for each feature originating from the centroid of the feature, the direction of which vector is also defined by Cartesian coordinates. The vector represents the optimal directionality of the feature, e.g., the direction of an optimal hydrogen bond for hydrogen-bonding features, inter alia. Preferably, the model comprises at least four features: 1 hydrogen bond donor, 1 hydrophobic feature and 2 ring aromatic features. In another embodiment, the model comprises at least five features: 3 hydrophobic features, 1 positive ionizable feature and 1 ring aromatic feature.
The coordinates of the model of the invention defines the relative relationship between the features, and therefore, those of skill in the art will recognize that the specific coordinates are dependent upon the specific coordinate system used, and thus, although rotation or translation of these coordinates may change the specific values of the x, y and z coordinates, the coordinates will define the claimed model. Those skilled in the art will also recognize that the model of the invention may encompass any model, after optimal superposition of the models, comprising the identified features and having a root mean square of equivalent features of less than about 3.0 xc3x85. More preferably, the model of the invention encompasses any model comprising the identified features and having a root mean square of equivalent features of less than about 1.5 xc3x85, and most preferably, less than about 1.0 xc3x85.
The present invention also relates to a method for identifying NK-1 antagonist compounds, which do not possess significant inhibitor potency towards CYP2D6, from structural and literature databases of experimental compounds comprising the use of the pharmacophore model generated in accordance with the methods of the present invention. In a preferred embodiment, an NK-1 antagonist compound which does not possess significant inhibitory potency towards CYP2D6 is identified by predicting the IC50 value of said compound. Preferably, the IC50 value for an NK-1 antagonist compound, which does not possess significant inhibitory potency towards CYP2D6, is greater than or equal to 1 xcexcM; more preferably, greater than or equal to 10 xcexcM; and most preferably, greater than or equal to 100 xcexcM.
The present invention also relates to NK-1 antagonist compounds, which do not possess significant inhibitory potency towards CYP2D6, identified by the methods of the present invention. Further, the present invention is directed to pharmaceutical compositions comprising an NK-1 antagonist compound that does not possess significant inhibitory potency towards CYP2D6 identified by the methods of the present invention.
The present invention further relates to methods of treatment for a condition, disorder or disease for which an NK-1 antagonist receptor compound is therapeutically useful comprising the administration of an NK-1 receptor antagonist compound that does not possess significant potency towards CYP2D6, a pharmaceutically acceptable derivative or pharmaceutical composition thereof, identified by a method of the present invention comprising the use of the pharmacophore model of the invention. The condition, disease or disorder may be selected from the group consisting of nausea, asthma, migraine, arthritis, post-operative pain and depression.
The present invention is also related to a method of designing de novo compounds that are NK-1 antagonist compounds which do not possess significant inhibitory potency towards CYP2D6 comprising the step of (i) correlating the two- and/or three-dimensional descriptors for a pharmacophore model for NK-1 antagonist compounds that possess significant inhibitory potency towards CYP2D6 with randomly generated molecules having chemical features corresponding to said descriptors; and (ii) choosing a generated molecule with a CYP2D6 IC50 of 1 xcexcM or greater. Preferably, the CYP2D6 inhibitory activity should correspond to an IC50 of 10 xcexcM or greater; more preferably 100 xcexcM or greater. The compounds having features corresponding to said descriptors may be randomly generated by any variety of computational methods from a library of known chemical features and conformational preferences of chemical groups and multiple chemical groupings.
The present invention is also related to a method of designing de novo NK-1 antagonist compounds which have selective inhibitory potency towards CYP2D6 comprising the step of
(i) choosing a target degree of CYP2D6 inhibitory potency;
(ii) generating a set of two- and/or three-dimensional descriptors for a pharmacophore model for NK-1 antagonist compounds that possess significant inhibitory potency towards CYP2D6 corresponding the inhibitory potency of step (i); and
(iii) correlating said descriptors of step (ii) with compounds having chemical features corresponding to said descriptors.
The present invention relates to a computer comprising a pharmacophore model for the inhibitory potency towards CYP2D6 of NK-1 antagonist compounds generated in accordance with the methods of the present invention. The present invention relates to a computer comprising a pharmacophore model for the CYP2D6 inhibitory potency of NK-1 antagonist compounds for use in the design or screening of a molecular structure having NK-1 receptor antagonist activity and CYP2D6 inhibitory activity.
The term xe2x80x9csignificant inhibitory potencyxe2x80x9d in the context of enzyme inhibition, unless otherwise indicated, refers to the ability of a compound over a characteristic concentration range to interfere with the function of said enzyme, whether permanently or temporarily, so as to deprive said enzyme of the ability to effect or participate in the transformation of chemical and/or biological substances.
The term xe2x80x9ctreatingxe2x80x9d refers to, and includes, reversing, alleviating, inhibiting the progress of, or preventing a disease, disorder or condition, or one or more symptoms thereof; and xe2x80x9ctreatmentxe2x80x9d and xe2x80x9ctherapeuticallyxe2x80x9d refer to the act of treating, as defined above.